1 Supercritical CO 2 extraction of oregano and clove buds mixture-synergistic effect and chemical composition of extract J. Ivanovic 1, *, I. Zizovic 1 , M. Ristic 2 , M. Stamenic 1 , D. Skala 1 1 Faculty of Technology and Metallurgy, Karnegijeva 4, 11000 Belgrade, Serbia 2 Institute for Medical Plant Research „Dr Josif Pančić“, Tadeuša Košćuška 1, 11000 Belgrade, Serbia [email protected]Abstract The goal of present study was to investigate kinetics of supercritical carbon dioxide (SC CO 2 ) extraction of essential oils from dried clove buds and oregano leaves. Total extract yields after exhaustion of plant material at 10 MPa and 40°C (after consumption of 94.3 kgCO 2 /kg plant material ) were 18.2% and 1.0 %, respectively. Synergistic effect in the extraction process was also investigated using mixture of clove buds and oregano leaves with initial mass ratio of clove buds to oregano leaves of 1:18.2. This mass ratio was applied assuming that it will give the extract with equivalent masses of clove and oregano extract (50:50) and that expected total yield will be 1.89 %. However, the experimentally determined total yields of extract in three repeated experiments were in the range of 2.10-2.66 % and it was higher than expected. The experimental data of extract yield versus CO 2 consumption were used to obtain the parameters of Sovová model and to propose logical interpretation of SFE extraction from oregano leaf and clove bud mixture. Chemical analyses of extracts were accomplished by GC/FID and GC/MS analytical methods. Extract obtained by SFE from mixture of oregano leaf and clove bud contains a lower amount of less and higher amount of heavier soluble monoterpene and sesquiterpene type compounds (eugenol, eugenol acetate and trans-caryophyllene) than expected on the basis of separate SFE from oregano and clove buds. Such results indicate that some synergistic effect in the SFE process really exists. Key words: Supercritical extraction; Oregano; Clove buds; Synergistic effect; Modelling. INTRODUCTION The clove (Syzygium aromaticum, syn. Eugenia aromaticum or Eugenia caryophyllata), is a tree of the family Myrtaceae, is indigenous to Indonesia, Madagascar, Sri Lanka and the south of China grown primarily for the unopened flower buds which are dried to produce the commercial product. Clove buds contain 15 to 21% by weight of volatile oil rich in eugenol (up to 95%), eugenol acetate and trans-caryophyllene. Clove oil and eugenol are well-known for antibacterial, antifungal, insecticidal and antioxidant properties. Eugenol is used as anti-microbial agent against oral bacteria in preparations for dental care and periodontal disease, while sesquiterpenes found in clove have been investigated as potential anticarcinogenic agents [1-3]. The essential oils from clove buds are traditionally obtained by steamdistillation and hydrodistillation [3, 4]. Clove extracts can be isolated by conventional extraction with organic solvents [3, 4], by sub-critical water extraction [4] and supercritical fluid extraction (SFE) [3-9].
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Supercritical CO2 extraction of oregano and clove buds
mixture-synergistic effect and chemical composition of
extract
J. Ivanovic1,
*, I. Zizovic1, M. Ristic
2, M. Stamenic
1, D. Skala
1
1 Faculty of Technology and Metallurgy, Karnegijeva 4, 11000 Belgrade, Serbia
2 Institute for Medical Plant Research „Dr Josif Pančić“, Tadeuša Košćuška 1, 11000
ID% 96.01 76.65 85.08 85.29 87.25 86.15 KI, Kovats index; RT/MS, retention time of corresponding constituent obtained by GC/MS; ID%, percent of given identified compounds
Similar composition profiles of the supercritical extracts from clove buds obtained within the
pressure range of 10-30 MPa and at temperatures of 30-55°C have been previously reported in
the literature (Table 3). The major compounds in the supercritical extracts of oregano were
monoterpene alcohols and their derivatives, carvacrol (56.51%), thymol (5.92%) and
thymoquinone (13.13%) (Table 2). In the previously published data on chemical composition
of supercritical extraction of oregano, the main carvacrol and thymol are the most abundant
components but their content oscillate due to different extraction conditions [14-16] and
origin of row material [15].
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Table 3. The composition profiles of supercritical extracts from clove buds and oregano leaves
Origin of plant material p (MPa);t(°C);time (h);
kgCO2/kg plant material Major compounds (mass %) References
Clove buds
Canary Islands 10 MPa; 40°C; 5.35 h;
94.3
Eugenol (65.43)
Eugenyl acetate (19.74)
β-Caryophyllene (12.84)
in this study
China
10-30 MPa;
30-50°C; 2 h
Optimal:
10 MPa; 50°C; 6160
Eugenol (58.77);
Eugenyl acetate (19.6) [3]
Madagascar 20 MPa; 55°C; 5.5 h; 55
Eugenol (16.42)
Eugenyl acetate (3.39);
β-Caryophyllene (1.73)
[4]
Italy
9 MPa; 50°C
(1st : 9 MPa; -10°C
2nd
: 1.5 MPa; 10°C);
10.5 h; 54
Eugenol (65.87);
Eugenyl acetate (19.0);
β-Caryophyllene (11.1)
[5, 6]
Italy 15 MPa; 25°C; 1.5 h; 51
Eugenol (79.4);
β-Caryophyllene (10.2);
α-Humulene (12.0);
Eugenyl acetate (9.2)
[7]
Brazil 10 MPa; 25°C; 2 h; 5.75
Eugenol (72.03);
Eugenyl acetate (15.61)
β-Caryophyllene (10.92);
α-Humulene (1.44)
[8]
Brazil 6.447-6.97 MPa; 10-
12°C;-
Eugenol (48.41-58.62);
Eugenyl acetate (19.50-28.19)
β-Caryophyllene (19.77-19.91);
α-Humulene (1.60-2.49)
[9]
Oregano leaves
Serbia 10 MPa; 40°C; 4.5 h;
94.3
Carvacrol (56.51);
Thymoquinone (13.13);
Thymol (5.92)
in this study
Estonia 17.2-25.5 MPa; 45°C;
2.5-7% ethanol; 1 h; -
Thymol (7.2-11.2);
Carvacrol (8.0-10.9);
Thymol acetate (0.1-12.4)
[14]
Turkey and Hungary
Fractional extraction:
8-30 MPa; 40°C
(1st: 8 MPa; 33-37°C
2nd
: 2 MPa; 20-25°C); 6-
7 h; 90-250
Turkish oregano:
Carvacrol (86.1-90.5);
p-Cymene (1.50-5.90);
Linalool (1.30-2.60)
Hungarian oregano:
Carvacrol (77.85);
Thymol (9.15)
[15]
Spain 12 MPa; 40-60°C; 0.42
h; -
Linalool (43.6);
Thymol (21.7);
Carvacrol (6.4)
[16]
As expected, the composition mixture of oregano and clove extracts obtained by separate SFE
(1:1) was similar to theoretically expected on the basis of the separate extractions from
oregano and clove. The extract obtained by simultaneous SFE from oregano and clove
mixture obtained in the first experiment comprised almost equally quantities of carvacrol and
eugenol unlike the extract obtained in the second experiment which comprised almost three
times higher content of eugenol. Since the experiment I had shown significant disagreement
with the Experiment II and Experiment III with respect to kinetic data and chemical
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composition analyses, it can not be regarded as representative. The increase of total extract’s
yield of 12.7 % is calculated as mean value from Experiment II and III and used for further
analyses.
The quantities of the main components in the representative extract obtained by
simultaneous SFE were compared to expected quantities of particular components in
accordance to chemical analysis of clove and oregano extracts obtained by separate SFEs.
Therewith, the higher amount of the main components of clove (eugenol, trans-caryophyllene
and eugenol acetate) and lower amount of the main components of oregano extract than
expected on the basis of the separate SFEs were observed (Table 4). An increase of eugenol,
trans-caryophyllene and eugenol acetate prevails over decrease of carvacrol, thymol and
thymoquinone and result in the increase of the total extract obtained after exhaustive
simultaneous SFE at given conditions.
Table 4. Quantitative analysis of the extracts
Components Clove
(mass%) Oregano
(mass%) Mixture
(mass%) Mass
(mg/100g
plant
material)
Mixture
(theoretical)
(mass%)
Mass
(mg/100g
plant
material)
Increase
(%)
Yield
(g/100g of plant material)
18.2 1.0 2.13 2.13 1.89 1.89 12.7
TQ - 13.13 1.39 29.61 6.56 124.00 -76
T - 5.92 0.51 10.86 2.96 55.90 -81
C - 56.51 17.46 371.90 28.26 534.11 -30
E 63.43 - 44.15 940.40 31.72 599.51 57
TC 12.84 0.99 8.20 174.66 6.92 130.79 33
EA 19.74 0.1 13.58 289.17 9.92 187.49 55
The evaluation of differential and cumulative yield (calculated with respect to mass of the
extract) of major compounds during simultaneous SFE from oregano and clove mixture at 10
MPa and 40°C with respect to SC CO2 consumption (kgCO2/kg plant material) (Fig. 3.) has been
made in order to study the dynamics of extraction of the major compounds present in the
extract. Differential yield of the carvacrol, thymol and thymoquinone (major components of
the oregano) gradually increases in the first and transitional extraction period of SFE (up to
consumption of 50 kgCO2/kgplant material or 3h). After 3 hours of extraction only negligible
quantity of these compounds was still extracted but not in expected quantities which might be
observed after their complete exhaustion. Unlike, the constant differential yield decrease was
observed for the extraction of eugenol, trans-caryophyllene and eugenyl acetate during the
extraction (Fig. 3a). Slower extraction of these components occurs since the most of the clove
oil remains in the intact secretory cells and cavities.
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Fig. 3. Differential and cumulative yield of major compounds during simultaneous SFE from mixture of clove
buds and oregano leaves (mass %)
Cumulative yields of some compounds shown in Fig. 3b remain almost constant during
extraction. It was not expected that slight increase of cumulative yields for oregano main
component (carvacrol) and decrease for clove main component (eugenol) could be result of
simultaneous extraction of clove and oregano. Namely, the fast extraction of compounds
which belong to oregano from the easily accessible fraction (carvacrol, thymol and
thymoquinone) is expected at the beginning of extraction, while an increase of cumulative
yield of other compounds from clove must prevail in the later stage of extraction. In the
previous studies, yield of eugenol during the clove buds SFE at similar extraction conditions
constantly increases with extraction time and starts to decrease after long time of extractions
(after 6.5 h) [4,6]. In this study, decrease of cumulative yield eugenol in the later extraction
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period during simultaneous SFE from the oregano and clove mixture can be result of
increased rate of extraction of eugenol from herbaceous matrix of clove in the presence of
carvacrol.
The following explanation for observed effect could be given. Due to applied pre-
treatment of the plant material, the most of the oregano extractible substances (carvacrol) are
easily accessible to SC CO2 and readily dissolved in it. During the exposure of the plant
material to SC CO2, supercritical fluid with already dissolved oregano components diffuse
into secretory cavities and cells of oregano. The readily dissolved carvacrol influences
solubility power of SC CO2 acting like co-solvent and promoting extraction of the heavier
soluble compounds such eugenol, eugenyl acetate and trans-caryophyllene. One part of the
oregano extract (carvacrol) probably stays in the finest secretory cavities while in the same
time increased quantity of eugenol, eugenol, eugenyl acetate and trans-caryophyllene is
dissolved in SC CO2 inside the matrix. The increased quantity of eugenol, eugenol, eugenyl
acetate and trans-caryophyllene now needs less time to diffuse from solid herbaceous matrix
into CO2 phase. This could explain by lower values of resistance to internal mass transfer
during simultaneous SFE of clove and oregano mixture with respect to SFE from clove buds
alone.
These assumptions still need to be confirmed by further investigations on
simultaneous SFE of the same system (oregano and clove) with various initial composition or
to use some other and similar mixture of plant material.
CONCLUSION
The exhaustive SFE extractions from oregano leaves and clove buds as well as
simultaneous extraction of mixture of this plant materials (10 MPa; 40 oC SC CO2) were
analyzed in this study. Extraction curves for pure plants or their mixture were analyzed using
model proposed by Sovová. Calculated mass transport phenomena and determined
parameters of model were used for description of the experimentally obtained curve for
simultaneous SFE from mixture. The results of chemical composition and eugenol and
carvacrol solubility data in the SC CO2 have also used for describing experimental data.
Presented results indicated that increase of the eugenol, trans-caryophyllene and
eugenyl acetate content in extract and decrease of the carvacrol, thymol and thymoquinone
content in the extract comparing to the expected and theoretical contents after exhaustion of
the mixture of clove bud and oregano could be result of decreased internal mass transfer
resistance caused by different rate of diffusion of eugenol in the SC CO2 in the presence of
carvacrol. Moreover, it is suggested that main components of oregano (primarily carvacrol
and thymol) change the solubility power of the SC CO2 in the first period of extraction (acting
like modifier of SC CO2) and thus increase the solubility of heavier and less soluble
compounds in the mixture (eugenol).
The present study reveals possibility of optimization of SFE process with respect to
extraction rate, as well as quantity and quality of extracts by co-extraction of plant materials
with different structure and chemical compositions.
REFERENCES
[1] Nurdjannah, N., Bermawie, N. Clove, In: Handbook of Herbs and Spices (Ed.) K.V. Peter,